Project Summary/Abstract Lung cancer is the leading cause of cancer-related death; lung adenocarcinoma (LUAD) is the most frequent type. The pathogenesis of LUAD is poorly understood. We recently discovered that sodium-glucose transporter 2 (SGLT2) is a previously unrecognized system of metabolic supply specifically active in early- stage LUAD and required for lung carcinogenesis. SGLT2 inhibition prolongs survival, delays cancer development, and slows tumor growth in genetically engineered murine models (GEMMs) and in patient- derived xenografts (PDXs) of LUAD. Pre-malignant lesions and early-stage LUADs express SGLT2 but not the well-known GLUT1 transporter, whereas more advanced tumors display heterogeneous expression of SGLT2 in low-grade and GLUT1 in high-grade areas of the same tumor. Why do tumors need to switch from SGLT2 to GLUT1 as they progress? Our data suggest that this switch is associated with a reprogramming of glucose metabolism: SGLT2+ tumors use oxidative glucose metabolism, GLUT1+ tumors use glycolysis. Glucose uptake can be studied in vivo by positron emission tomography (PET) with 2-[18F] fluorodeoxyglucose (FDG), transported by GLUTs and not SGLTs, and methyl 4-[18F]fluorodeoxyglucose (Me4FDG), specific for SGLTs. We will investigate the heterogeneity of glucose transport in LUAD, to understand the biological significance and the mechanisms that regulate the expression of SGLT2 and GLUT1. In Aim 1, we will characterize glucose metabolism in Me4FDG+ versus FDG+ tumors in GEMMs and in PDXs by PET imaging, respirometry, metabolomics, metabolic tracing, histology, and transcriptomics, with or without CRISPR knockout or overexpression of SGLT2 and GLUT1. In Aim 2, we will investigate the role of GSK3 kinase, which we identified as an SGLT2 regulator, in the regulation of metabolic/differentiation programs associated with the switch from SGLT2 to GLUT1. In Aim 3, we will investigate the role of hypoxia in the switch from SGLT2 to GLUT1 expression as LUAD progresses from well- to poorly differentiated, including correlation between FDG or Me4FDG uptake with markers of hypoxia (F-MISO, pimonidazole), and evaluation of the effect of hypoxia- inducible factors knockout on SGLT- and GLUT-dependent uptake. The goal of the proposed research is the elucidation of a novel mechanism of metabolic reprogramming in cancer: switching between two different glucose transporters, cancer cells can redirect the metabolic fate of glucose towards different pathways; this observation has important implications as interfering with the Warburg effect or with glucose transport has been proposed as a novel therapy for cancer; the ability of cancer cells to change their metabolism by switching glucose transporters is a potentially targetable mechanism of resistance, as there are specific inhibitors of both SGLT2 and GLUT. Moreover, the availability of PET tracers that can selectively measure SGLT or GLUT activity in vivo (Me4FDG and FDG, respectively) is a promising tool for characterizing the metabolic phenotype of cancers in vivo, in order to predict their response to treatments.